This invention relates to methods for preparing acrylamide gels for electrophoretic analysis, and more particularly to methods for producing acrylamide gel solutions and then forming these solutions into polyacrylamide gel samples for electrophoretic analysis.
Polyacrylamide gel electrophoresis (PAGE) is a powerful technique commonly used in biochemistry and biomedicine research. Depending on the purpose, this technique can be used to perform either analytical or preparative studies of protein and nucleic acid. A book (article) entitled "SDS Polyacrylamide Gel Electrophoresis of Proteins" by B. J. Smith, relates to PAGE. The following are some of the common applications of PAGE which are used in protein chemistry and molecular biology:
1. Protein molecular weight determination.
2. Monitoring protein purification.
3. Preparative protein isolation.
4. Nucleic acid separation.
5. DNA sequencing.
PAGE was first introduced to the field of protein analysis more than twenty years ago. The most widely used PAGE procedure is that of Laemmli (1970). Electrophoretic separation of biological molecules on polyacrylamide gel is based on the net charge, shape and size of these molecules. These gels are typically formed by the polymerization of an acrylamide monomer solution. The process is initiated by adding a free radical-generating compound into the acrylamide solution. Gels are made with different concentration of acrylamide and cross linked by adding initiator (such as ammonium persulfate) and an accelerator, for example, tetramethyl-ethylenediamine (TEMED). A detergent, such as sodium dodecyl sulfate (SDS), or urea are incorporated into the gel if samples are analyzed under dissociation conditions.
Probably the most widely used of techniques for analyzing mixtures of proteins is SDS polyacrylamide gel electrophoresis. In this technique, proteins are reacted with the anionic detergent SDS, or sodium lauryl sulfate, to form negatively charged complexes. The amount of SDS bound by a protein, and so the charge on the complex, is roughly proportional to its size. Commonly, about 1.4 g. SDS is bound per 1 g. protein, although there are exceptions to this rule. The proteins are generally denatured and solubilized by their binding of SDS, and the complex forms a prolate ellipsoid or rod of a length roughly proportionate to the protein's molecular weight. Thus, proteins of either acidic or basic form negatively charged complexes that can be separated on the bases of difference in sizes by electrophoresis through a sieve-like matrix of polyacrylamide gel.
SDS-polyacrylamide gel electrophoresis is reproducible, versatile, and convenient. It uses either a tubular disc gel or a slab gel. The slab gel technique is an improvement of the disc gel electrophoresis. Less gel and sample materials are required per assay on a slab gel system, and the resulting gel can be easily used for further analysis. Electrophoresis on slab gel of full size (.about.16-20.times.16 cm), or mini-size (.about.10.times.10 cm) of 1.5 mm, 1.0 mm, 0.75 mm and even 0.5 mm thickness, is made with composition described by Davis (1964) and by Laemmli. The gel is typically prepared at an acrylamide concentration of 15%, 12%, 10%, 8% or 7.5% or of a gradient in concentration ranging from 5% to 20%. Often, a 4% stacking gel is casted on the top portion of the gel to facilitate good separations.
The conventional method to make slab polyacrylamide gel is by casting a polymeric gel between a pair of glass plates. Thus, the initiator-containing acrylamide gel solution is cast in the rectangular cavity formed between the two glass plates. The thickness of the gel is determined by the thickness of spacers inserted between the glass plates which form the cavity therebetween. The assembly uses two or three spacers made of plastic strips. These spacers can be 1 cm wide and 1.5, 1.0, 0.75 and 0.5 mm thick. Two of them serve as side spacers and are laid on the sides of glass plates. They have the same length as the height of longer bottom plate. If a third spacer is used, it is positioned below the side spacers on the bottom of the glass plate and is a little longer than the width of the plate. The whole set-up is held in place with either clamps or specially made holders. Warm agar solution, tape, or special gel casting stand are some of the ways to seal any possible leakage. Acrylamide gel solution with TEMED added is poured into the space between glass plates. The gel forms after the acrylamide polymerization is completed. The bottom spacer portion is then removed from the assembly. To carry out electrophoresis, the lower part of the gel sits in a buffer bath and the top part of the gel is enclosed by a top buffer bath. Then, current is applied to the system to separate molecules of protein or nucleic acid.
There are, however, a number of disadvantages to the above-described conventional process. First, assembling plates and spacers for casting polyacrylamide gel is a cumbersome and time consuming process. Even with a special casting stand, a certain degree of manipulation is required in order to put the assembly together. Second, the cost of the electrophoresis system including a gel casting system is expensive, and parts from different system are not interchangeable. Finally, sealing between plastic strips and glass plates is unpredictable. Besides, the junction between side spacers and bottom spacer, or the casting stand has a gap that is very difficult to close. Acrylamide solution often leaks from within the cavity while waiting for gel to polymerize. Attempts to mend leaks during gelling often ends up with unsatisfactory electrophoresis results.
The chemical composition of the gel varies depending on specificity of experiments. Polyacrylamide gel electrophoresis is divided into denatured, partially denatured and non-denatured PAGE categories. Denatured PAGE system is used for protein and nucleic acid studies. For protein molecular weight determinations, the gel and the electrophoresis buffer include a detergent (SDS). In this way, the intra-molecular disulfide bonds of sample proteins are reduced before electrophoresis. In protein chemistry, PAGE in the presence of detergent SDS separates mixture of protein based on the molecular weight. During separation, while applying electrical current, diffusion through gel is minimized. Molecules resolve into narrow bands depend on their mobility in the gel matrix. Protein bands are visualized after staining. The gel is dried for preservation or to continue with other analysis.
The denatured PAGE system is also used in nucleic acid and gene sequencing studies. The sequencing gel contains urea and/or foramide. After electrophoresis separation, radioactive labeled nucleotides are examined under UV light. The gel is also exposed to X-ray film for establishing a permanent record.
A partially denatured PAGE system uses a detergent-containing gel and buffer to assay non-denatured, native proteins. For non-denatured PAGE systems, neither gel nor buffer contains detergent. This is used to assay native proteins.
Most laboratories follow the conventional method for making their own gel (as previously described). All stock solutions except 10% ammonium persulfate are prepared and stored for one to six months. Powdered chemicals, such as acrylamide, bis-acrylamide, tris base, HCl, SDS, ammonium persulfate, TEMED, urea, EDTA, boric acid and foramide, are available commercially for gel-making.
To make the gels described above in conventional way, laboratory personnel have to prepare several stock solutions. Several chemicals used in gel preparation are hazardous and require special cautions during handling. All chemicals used in gel mixture have to be at the highest purity, otherwise, aberrant electrophoresis results may occur. Furthermore, stock solutions take up space from cold storage facility such as refrigerator. On the day of preparing gels, the following procedure takes place:
1). A fresh 10% ammonium persulfate solution is prepared. PA1 2). Precise volumes of five stock solutions are measured out. PA1 3). The solution is mixed and then deaerated under vacuum or aspirator.
Several commercial products such as pre-mixed acrylamide powder, pre-mixed acrylamide solutions, and pre-cast mini-gel are designed to simplify the gel making process. However there are certain drawbacks related to each of the above mentioned products. Premixed powder do not eliminate the potential danger of handling bio-hazardous chemicals. Premixed acrylamide solution at specific gel strength limits the application of electrophoresis. Premixed stock acrylamide solutions are supplied in large volumes (500 ml or more), which does not relief the problem of cold storage space. Limited shelf life of premixed solution means unavoidable wastes. In any case, the fresh 10% ammonium persulfate solution has to be made the day of casting the gel. Pre-cast mini-gel provides convenience for electrophoresis at high cost with a short shelf life and requirement of special storage.
Consequently, a need exists for method of preparing polyacrylamide gel samples for electrophoresis analysis which includes a polyacrylamide formation system which eliminates the potential danger of handling bio-hazardous chemicals, which incorporates the effective and efficient use of premixed chemicals for preparing the acrylamide solution at specific gel strength and without excessive waste, which relieves the problem of cold storage space, and a spacer system for casting polyacrylamide gel which requires a minimum degree of manipulation for assembly together, which effectively seals the two glass plates against leaking, which provides a predetermined thickness of polyacrylamide gel, and which consistently provides satisfactory electrophoresis results.